1. Field of the Invention
The present invention generally relates to the identification and use of diagnostic markers for cerebral injury. In a various aspects, the present invention particularly relates to methods for (1) the early detection and differentiation of secondary brain edema; (2) early growth of intracerebral hemorrhage (ICH); and (3) to identify patients who could benefit from aggressive therapies such as decompressive hemicraniectomy or hypothermia.
2. Background of the Invention
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
A stroke is a sudden interruption in the blood supply of the brain. Most strokes are caused by an abrupt blockage of arteries leading to the brain (ischemic stroke). Other strokes are caused by bleeding into brain tissue when a blood vessel bursts (hemorrhagic stroke). Because stroke occurs rapidly and requires immediate treatment, stroke is also called a brain attack. When the symptoms of a stroke last only a short time (less than an hour), this is called a transient ischemic attack (TIA) or mini-stroke. Stroke has many consequences.
The effects of a stroke depend on which part of the brain is injured, and how severely it is injured. A stroke may cause sudden weakness, loss of sensation, or difficulty with speaking, seeing, or walking. Since different parts of the brain control different areas and functions, it is usually the area immediately surrounding the stroke that is affected. Sometimes people with stroke have a headache, but stroke can also be completely painless. It is very important to recognize the warning signs of stroke and to get immediate medical attention if they occur.
Stroke or brain attack is a sudden problem affecting the blood vessels of the brain. There are several types of stroke, and each type has different causes. The three main types of stroke are listed below.
Ischemic stroke is the most common-type of stroke—accounting for almost 80% of strokes—and is caused by a clot or other blockage within an artery leading to the brain.
Intracerebral hemorrhage (ICH) is a type stroke caused by the sudden rupture of an artery within the brain. Blood is then released into the brain, compressing brain structures.
Subarachnoid hemorrhage is also a type of stroke caused by the sudden rupture of an artery. A subarachnoid hemorrhage differs from an intracerebral hemorrhage in that the location of the rupture leads to blood filling the space surrounding the brain rather than inside of it.
ICH causes a 35% to 50% 30-day mortality. Half of this mortality occurs within the first 2 days as a result of brain herniation, mainly caused by the continued bleeding that provokes an enlargement of the hematoma during the first 24 hours (Kazui S, Naritomi H, Yamamoto H, Sawada T, Yamaguchi T. Enlargement of spontaneous intracerebral hemorrhage. Incidence and time course. Stroke. 1996; 27:1783-1787.). Early hematoma growth (EHG) has been associated with early neurological worsening and poor outcome, but no clinical or radiological predictive factors have been identified and the pathogenesis remains unclear (see for instance Brott T, Broderick J, Kothari R, Barsan W, Tomsick T, Sauerbeck L, Spilker J, Duldner J, Khouri J. Early hemorrhage growth in patients with intracerebral hemorrhage. Stroke. 1997; 28:1-5; Fujii Y, Tanaka R, Takeuchi S, Koike T, Minakawa T, Sasaki O. Hematoma enlargement in spontaneous intracerebral hemorrhage. J Neurosyrg. 1994; 80:51-57.).
The pathophysiology of brain edema and secondary neuronal injury in ICH is as follows. After the initial arterial rupture, the activation of the coagulation cascade produces a large quantity of thrombin that is implicated in several functions, including chemotaxis of leukocytes, expression of adhesion molecules, release of inflammatory cytokines, blood-brain barrier disruption, and local metalloproteinase generation (see for instance Xi G et al 1998; Lee K R, Colon G P, Betz A L, Keep R F, Kim S, Hoff J T. Edema from intracerebral hemorrhage: the role of thrombin. J. Neurosurg. 1996; 84:91-96.). Furthermore, the release of iron after erythrocyte lysis may contribute to blood-brain barrier dysfunction, possibly through a free radical-mediated damage of endothelial wall (see for instance Xi G, Hua Y, Bhasin R R, Ennis S R, Keep R F, Hoff J T. Mechanisms of edema formation after intracerebral hemorrhage. Effects of extravasated red blood cells on blood flow and blood-brain barrier integrity. Stroke. 2001; 32:2932-2938.). Although all these mechanisms seem to be involved in edema formation after ICH (see for instance Castillo et al 2002; Abilleira S, Montaner J, Molina C, Monasterio J, Castillo J, Alvarez-Sabý'n J. Matrix metalloproteinase-9 concentration alter spontaneous intracerebral hemorrhage. J Neurosurg. 2003; 99:65-70.), their role in the EHG remains unclear.
EHG has been related to multifocal bleeding in the periphery of the clot caused by the rupture of arterioles and venules in the perilesional low-flow zone. Secondary brain injury has been attributed to ischemic damage and particularly to the toxic effects of thrombin generation by the clot (see for example Mendelow A D. Mechanisms of ischemic brain damage with intracerebral hemorrgae. Stroke. 1993; 24(suppl 1):115-117.). In experimental ICH, thrombin activates the inflammatory cascade and the expression of matrix metalloproteinases (MMPs), causing the breakdown of the blood-brain barrier and edema formation (see for example Xi G, Wagner K R, Keep R F, Hua Y, de Courten-Mayers G, Broderick J P, Brott T G, Hoff J T. Role of blood clot formation on early edema development after experimental intracerebral hemorrhage. Stroke. 1998; 29:2580-2586; Rosenberg G A, Navratil M. Metalloproteinase inhibition blocks edema in intracerebral hemorrhage in the rat. Neurology. 1997; 48: 921-926.). In this context, high serum concentrations of cytokines and MMP-9 have been associated with a large volume of peripheral hypodensity in human ICH (see for example Castillo J, Davalos A, Alvarez-Sabin J, Pumar J M, Leira R, Silva Y, Montaner J, Kase C S. Molecular signatures of brain injury after intracerebral hemorrhage. Neurology. 2002; 58:624-629.). MMPs are able to degrade the basal membrane components, such as cellular fibronectin (c-Fn), a glycoprotein especially important for the adhesion of platelets to fibrin, a function necessary for the blockade of bleeding.
The knowledge of the underlying mechanisms and factors associated with EHG is crucial because they represent potential targets for therapeutic interventions. In the only previous prospective study, Brott et al. failed to reveal any clinical, radiological, or analytic predictor of ICH growth (Brott T, Broderick J, Kothari R, Barsan W, Tomsick T, Sauerbeck L, Spilker J, Duldner J, Khouri J. Early hemorrhage growth in patients with intracerebral hemorrhage. Stroke. 1997; 28: 1-5.).
Massive middle cerebral artery (MCA) infarction accounts for 10% to 15% of all MCA infarctions, and of these patients, malignant MCA (m-MCA) reaches 40% to 50%. The syndrome of m-MCA infarction, which is attributable to brain edema, is more frequent in younger patients and has a poor prognosis both short and long term. In 80% of patients, it leads to death, and those patients who survive experience severe neurological deficits.
Conservative treatments fail to improve mortality and disability. Early hemicraniectomy and hypothermia are feasible and have been proposed as effective treatments for this condition because they change the natural history of the disease.5 However, those patients who will develop m-MCA syndrome are currently not revealed by clinical, neuroimaging, or biochemical markers sufficiently early and with sufficient accuracy as to indicate an aggressive management.
The loss of integrity of the endothelial basal lamina is believed to be the primary cause of edema after focal cerebral ischemia. Matrix metalloproteinase-9 (MMP-9), a proteolytic zinc-dependent enzyme for which expression is increased during stroke (for example see Clark A W, Krekoski C A, Bou S S, Chapman K R, Edwards D R. Increased gelatinase A (MMP-2) and gelatinase B (MMP-9) activities in human brain after focal ischemia. Neurosci Lett. 1997; 238:53-56.), and in experimental models of focal ischemia, 9 it degrades the endothelial basal lamina10 and plays an essential role in producing edema and hemorrhagic transformation (for example see Hoe Heo J, Lucero J, Abumiya T, Koziol J A, Copeland B R, del Zoppo G J. Matrix metalloproteinases increase very early during experimental focal cerebral ischemia. J Cereb Blood Flow Metab. 1999; 19:624-633; Rosenberg G A, Mun-Bryce S, Wesley M, Kornfeld M. Collagenase induced intracerebral hemorrhage in rats. Stroke. 1990; 21:801-807.).
In a recent study, serum cellular-fibronectin (c-Fn), a component of the basal lamina, was shown to be a more accurate predictor of hemorrhagic transformation than MMP-9 in acute ischemic stroke patients treated with tissue plasminogen activator (tPA). (For example see Castellanos M, Leira R, Serena J, Blanco M, Pedraza S, Castillo J, Davalos A. Plasma cellular-fibronectin concentration predicts hemorrhagic transformation after thrombolytic therapy in acute ischemic stroke. Stroke. 2004; 35:1671-1676.) Therefore, an increased expression of blood-brain barrier (BBB) disruption markers in cerebral ischemia may partially explain the syndrome of m-MCA infarction. The instant invention shows the association between plasma concentrations of MMP-9, c-Fn, excitatory amino acids (EAAs), and inflammatory molecules with the development of brain edema and subsequent m-MCA syndrome in patients with complete MCA infarction.
Results for mortality rate and functional outcome after hemicraniectomy in massive MCA infarction have been contradictory (for example see Schwab S, Steiner T, Aschoff A, Schwarz S, Steiner H H, Jansen O, Hacke W. Early hemicraniectomy in patients with complete middle cerebral artery infarction. Stroke. 1998; 29:1888-1893; Morley N C D, Berge E, Cruz-Flores S, Whittle I R. Surgical decompression for cerebral oedema in acute ischaemic stroke (Cochrane Review). In the Cochrane Library. 2003, Issue 3. Oxford, UK: Update Software; 2003.). This might be explained by the lack of reliable predictors of m-MCA infarction. Studies into the value of neuroimaging and clinical and biochemical markers of malignant brain edema have found few predictors to be sufficiently sensitive and specific as to be useful in clinical practice (Table 5). Clinical factors alone are not sufficient to identify patients with impending brain edema (for example see Kasner S E, Demchuk A M, Berrouschot J, Schmutzhard E, Harms L, Verro P, Chalela J A, Abbur R, McGrade H, Christou I, Krieger D W. Predictors of fatal brain edema in massive hemispheric ischemic stroke. Stroke. 2001; 32:2117-2123; Krieger D W, Demchuk A M, Kasner S E, Jauss M, Hantson L. Early clinical and radiological predictors of fatal brain swelling in ischemic stroke. Stroke. 1999; 30:287-292.). CT scan showed acceptable sensitivity in some studies but low specificity in identifying candidates for hemicraniectomy (for example see von Kummer R, Meyding-Lamade U, Forsting M, Rosin L, Rieke K, Hacke W, Sartor K Sensitivity and prognostic value of early CT in occlusion of the middle cerebral artery trunk. AJNR Am J Neuroradiol. 1994; 15:9-15; Berrouschot J, Sterker M, Bettin S, Koster J, Schneider D. Mortality of space-occupying (“malignant”) middle cerebral artery infarction under conservative intensive care. Intensive Care Med. 1998; 24:620-623.). A recent approach has been to monitor biochemical markers and intracranial pressure (ICP) using a microdialysis probe inserted into the brain tissue (for example see Dohmen C, Bosche B, Graf R, Staub F, Kracht L, Sobesky J, Neveling M, Brinker G, Heiss W-D. Prediction of malignant course in MCA infarction by PET and microdialysis. Stroke. 2003;34:2152-2158.). However, this technique is complex, not widely available, invasive, and did not predict fatal outcome early enough for the successful implementation of invasive therapies because clinical deterioration often preceded the appearance of the analyzed biochemical markers and increased ICP. Promising results have been obtained with recent neuroimaging tests such as single-photon emission CT (for example see Berrouschot J et al. 1998), positron emission tomography of C-flumazenil (for example see Dohmen C et al. 2003), and diffusion-weighted MRI (for example see Oppenheim C, Samson Y, Manai R, Lalam T, Vandamme X, Crozier S, Srour A, Cornu P, Dormont D, Rancurel G, Marsault C. Prediction of malignant middle cerebral artery infarction by diffusion-weighted imaging. Stroke. 2000; 31:2175-2181. Thomalla G J, Kucinski T, Schoder V, Fiehler J, Knab R, Zeumer H, Weiller C, Rother J. Prediction of malignant middle cerebral artery infarction by early perfusion and diffusion-weighted magnetic resonance imaging. Stroke. 2003; 34:1892-1899.) in the prediction of m-MCA infarction within the suggested time window for hemicraniectomy. However these techniques evaluate infarct volume, the most reliable predictor of m-MCA, quickly and accurately but are unable to predict the development of massive brain edema directly as well as being very expensive as compared to the instant invention.
Accordingly, there is a present need in the art for a rapid, sensitive and specific differential diagnostic assay for the early detection and differentiation of EHG, m-MCA, and to identify patients who could benefit from aggressive therapies such as decompressive hemicraniectomy or hypothermia. Such a diagnostic assay would greatly increase the number of patients that can receive beneficial stroke treatment and therapy and in so doing reduce the costs associated with incorrect stroke diagnosis. Some content of this patent application was first published in the journal Stroke in its May 5 and Aug. 11, 2005, electronic issues, and thus we claim priority from these dates as well as the aforementioned dates.